Type II diabetes is simply characterized by hyperglycemia but is a disease state with a complex etiology. In addition to decreased glucose uptake by adipose and skeletal muscle, chronic hepatic glucose production contributes to the manifestation of this disease and is the central health related problem addressed by the proposed studies. Glucagon initiates the gluconeogenic program by activating the cAMP-signaling cascade that is, in part, mediated by the nuclear translocation of the CREB coactivator, TORC2. Indeed, the redistribution of TORC2 is a key regulatory step in glucose homeostasis and small molecules that are capable of blocking nuclear import of TORC2, or that facilitate its nuclear export, are likely to regulate hepatic glucose production. The goal of this project is to develop and validate a TORC2 subcellular localization assay that is amenable to high-throughput screening of large chemical libraries, with a long-term goal of identifying small molecules that target TORC2 redistribution and regulate hepatic gluconeogenesis. To this end, a portfolio of confirmatory screens will also be developed in parallel, which will discriminate small molecules producing false- positive results during the primary screening campaign. Finally, a cell-based gluconeogenesis assay will also be developed to validate the physiological relevance of small molecules hits coming from our primary and secondary screens. Collectively, the assays developed in the proposed studies will provide the toolsets necessary for a high-throughput screening campaign, to fulfill the long-term goal of identifying small molecule chemical probes that will aid in our understanding of aberrant gluconeogenesis, and that may lead to novel therapeutic molecules for the treatment of type II diabetes. PUBLIC HEALTH RELEVANCE: Characterized by high blood glucose, type II diabetes has reached pandemic proportions with approximately 20 million individuals affected in the United States alone. Contributing to the manifestation of this disease, chronic glucose production by the liver is one component of diabetes that is mediated by the cAMP-signaling pathway. By search for small molecules capable of blocking this signaling pathway we will uncover chemical probes that will facilitate our understanding this disease and will possibly function as novel therapeutic molecules for the treatment of individuals suffering from diabetes.